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7.
Membrane Filtration Method
 Pr ocedur e:

Open the filtration assembly and put
the filter paper aseptically by the
help of incinerated forcep on the
filtration gauge. Fix the filtration cup
again to rejoin filtration assembly.

Remove the cotton plug from flask
with your left hand’s last two finger
and let remain plug in your hand.
Pour all treated sample in the
filtration cup. Wait until all sample is
filter.

8.
Membrane Filtration Method
 Pr ocedur e:


Open the filtration assembly and
remove the filter paper aseptically
by the help of incinerated forcep
from the filtration gauge after all
sample is filtered. Rejoin the
filtration assembly again.
Adequately open the led of RODAC
plate and put the wet filter paper on
media containing plate (printed side
up) with incinerated forcep. Press
gentle and close the lid and invert
the plate.

10.
Pour Plate Method

Pr ocedure:




Take the treated sample.
Remove cotton plug with your right hand’s last two
finger, take up the flask and take 1 ml sample by
micropipette with your right hand too. After taking
sample plug the flask again and adequately open
the lid of petri plate with your left hand’s finger and
toe and then pour the sample in the plate.
Now Remove the cotton plug of media containing
flask as mention before and pour approx. 12 ml of
media inside the plate cavity and lid the plate.
Rotate the plate anti-clockwise and clockwise gently
so the media mixed well with 1 ml of sample. Be
precautious that media should not touch the lid of
petri plate.

11.
Pour Plate Method




Let the plate solidify. After solidification invert all
the petri plate.
Incubate all the petri plate in inverted position
for 72 hrs. at 22-25ºC and later 24 hrs. at 3035ºC.
Observe the petri plate at regular interval of
time. After incubation complete then count the
colonies with digital colony counter under SOP.
Record the colony count and document in
report of product.

12.
Spread Plate Method

Pr ocedure:




Take the pre-treated sample.
Take the solidify media containing plate.
Open the cotton plug of the flask with your right hand
and procure 1 ml of sample form the flask with the
help of micropipette and then plug the flask again.
Adequately open the led of media containing plate
and pour the sample and then close the led of plate.
Put down the micropipette and pick up the spreader
and put the plate at rotating plate, open it with left
hand and apply gently the spreader on the surface
of plate with right hand. Remain it touch with surface
until all sample spread well.

13.
Spread Plate Method





Let the plate dry from surface for 1 min.
After dry invert all the petri plate.
Incubate all the petri plate in inverted position
for 72 hrs. at 22-25ºC and later 24 hrs. at 3035ºC.
Observe the petri plate at regular interval of
time. After incubation complete then count the
colonies with digital colony counter under SOP.
Record the colony count and documented in
report of product.

14.
Serial Dilution Method
 Pr ocedur e:




Take the pre-treated sample.
Take the six test tube each containing 9 ml
saline solution.
Mark them 10-1 to 10-6.
Open the cotton plug of the flask with your right
hand and procure 1 ml of sample form the flask
with the help of micropipette and then plug the
flask again. Adequately unplug the test tube
and pour the sample in 10-1 saline tube
containing 9 ml saline and then close the led of
plate.

16.
Serial Dilution Method



Incubate all the petri plate in inverted position
for 72 hrs. at 22-25ºC and later 24 hrs. at 3035ºC.
Observe the petri plate at regular interval of
time. After incubation complete then count the
colonies with digital colony counter under SOP.
Record the colony count and documented in
report of product.

21.
Escherichia coli
 These
include identification of E. c o li, bacteria
pathogenic to human body and causes
infection of stomach. It is detected by using
specific, differential media which support
growth of only E. c o li.
 Primary test:


Pipette 1 ml of enrichment culture into tubes
containing 5 ml Mac Conkey’s broth and
incubate at 36-38 ˚C for 48 hrs.
If the content shows acid and gas carry out the
secondary test.

22.
Escherichia coli
 Secondary test:


After incubation, if the tube shows presence of
acid and gas, transfer 0.1 ml from tube to each
of two tubes containing, 5 ml Mac Conkey’s
broth and other containing 5 ml peptone water
and Incubate broth tubes in a water bath at
43.5˚C to 44.5˚C for 24 hrs. After incubation
examine tube (a) for acid and gas (b) for indole.
If the tubes shows turbidity then one loop full
culture is streaked over EMB and MCA and
incubate them for 24 hrs. at 43.5˚C to 44.5˚C.

23.
Escherichia coli
 tertiary test:


To test for indole production add 0.5 ml of
kovac’s reagent, shake well and allow to stand
for 1 min., if a red color is observed in the
reagent layer, indole is present,
The presence of acid and gas and of indole
indicates presence of Es c he ric hia c o li.

25.
Staphylococcus aureus
 Confirmatory
 Transfer
test: (coagulase test)
representative suspect colonies from the
agar surface of mannitol salt agar medium or
Vogel Johnson agar medium to individual tubes,
each containing 0.5 ml of mammalian, preferably
rabbit or horse plasma with or without additives.
Incubate in water bath at 37˚C examining the
tubes after 24 hrs.
 If coagulation in any degree is observed, the test
is positive.

26.
Pseudomonas aeruginosa

The identification or detection of pathogenic
bacteria which causes infections to the human
body can be identified by using specific differential
media which support only the growth of
Ps e ud o m o na s a e rug ino s a .
 Primary test:
Pretreat the preparation as described above.
 Place the prescribed quantity in a sterile screw
caped jar containing 100 ml of soybean casein
digest medium and incubate at 35-37˚C for 24 to 48
hrs.
 Observe the medium for growth.
 If any growth is observed, subculture a portion of
medium on a plate containing a layer of Cetrimide
Agar and incubate 35-37˚C for 18 to 24 hrs.
 If none of the plate contains colonies having
characteristics given in the table, carry out the


27.
Pseudomonas aeruginosa

Confirmatory test: (oxidase and pigment test)
 Streak representative suspect colonies from the agar surface of
cetrimide agar medium on the agar surface of pseudomonas agar
medium for detection of fluorescein and pseudomonas agar
medium for detection of pyocyanin contained in petri dishes.
 Cover and invert the inoculated media, and incubate at 35±2 ˚C for
not less than three days.
 Examine the streaked surface under UV light.
 Examine the plate to determine the whether colonies having the
characteristics, given in table, are present.
 Confirm any suspect colonial growth on one or more of the media
as Ps e ud o m o na s a e rug ino s a by means of oxidase test.
 Upon the colonial growth place or transfer colonies to strip or disks
of filter papers that previously has been impregnated with N, N, N,
N,-tetra-methyl, 4 phenyl adenine; if there is no development of
pink color, changing to purple, the specimen meets the
requirement of the test for the absence of Ps e ud o m o na s a e rug ino s a .
 The presence of Ps e ud o m o na s a e rug ino s a may be confirmed by other